Sage Journals: Discover world-class research

Abstract

One hundred and twenty five years ago, Roy and Sherrington made the seminal observation that neuronal stimulation evokes an increase in cerebral blood flow.¹ Since this discovery, researchers have attempted to uncover how the cells of the neurovascular unit—neurons, astrocytes, vascular smooth muscle cells, vascular endothelial cells and pericytes—coordinate their activity to control this phenomenon. Recent work has revealed that ionic fluxes through a diverse array of ion channel species allow the cells of the neurovascular unit to engage in multicellular signaling processes that dictate local hemodynamics.

In this review we center our discussion on two major themes: (1) the roles of ion channels in the dynamic modulation of parenchymal arteriole smooth muscle membrane potential, which is central to the control of arteriolar diameter and therefore must be harnessed to permit changes in downstream cerebral blood flow, and (2) the striking similarities in the ion channel complements employed in astrocytic endfeet and endothelial cells, enabling dual control of smooth muscle from either side of the blood–brain barrier. We conclude with a discussion of the emerging roles of pericyte and capillary endothelial cell ion channels in neurovascular coupling, which will provide fertile ground for future breakthroughs in the field.

Keywords

Ion channels calcium signaling calcium channels potassium channels transient receptor potential channels neurovascular coupling functional hyperemia cerebral blood flow neurovascular unit parenchymal arteriole pial artery smooth muscle endothelium astrocytic endfoot cerebrovascular resistance

Introduction

To support ongoing function, neurons must extract oxygen and glucose from the cerebral circulation on an as-needed basis. The continuous supply of nutrients to match local demand is made possible by neurovascular coupling (NVC). The NVC signaling cascade recruits multiple cell types to link neuronal activity to a rise in local blood flow—a phenomenon termed functional hyperemia—thus providing the energy substrates required to satisfy the enhanced metabolic requirements of active neurons. A number of mechanisms have evolved in parallel to ensure the fidelity of neurovascular communication, and many of these are dependent on ion channel signaling. Ion channels are also important contributors to the control of basal cerebral arteriolar tone, which sets the resting level of brain perfusion and permits dynamic changes in blood flow to match variations in local neuronal activity.

If the NVC cascade is impaired or cerebrovascular tone is pathologically altered, cerebral blood flow is compromised and neuronal dysfunction ensues. Cerebral blood flow is disturbed in a range of disorders, including Alzheimer’s disease,² hypertension,³ stroke,⁴ diabetes,⁵ and CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy).⁶ Thus, a fuller understanding of how cerebrovascular tone and functional hyperemia are controlled may unearth new treatment options for brain disorders with a vascular component.

In this review, our primary focus is on neurovascular ion channel signaling at the level of the parenchymal arteriole, where the influence of signaling molecules released from astrocytic endfeet on arteriolar diameter and blood flow is well established_. We explore two major themes. First, we examine the ion channels that facilitate tight control of smooth muscle (SM) membrane potential (V_m) and thus the contractile state (tone) of these cells. Second, we focus on the marked similarity in ion channel expression and signaling between astrocytic endfeet and endothelial cells (ECs), which exert dual control of SM tone from parenchymal and luminal sides of the vessel wall, respectively. Our discussion centers on studies examining ion channel signaling in vivo and in acutely isolated, intact ex vivo preparations. Where relevant, we compare parenchymal arterioles with vessels from other circulatory beds. We conclude with an exploration of a growing frontier of research: the control of cerebral blood flow at the capillary level. Here we highlight the known roles of ion channels in pericytes and capillary ECs, which likely have an important role in conducting signals from deep within the capillary bed upstream to parenchymal arterioles. The major ion channels reviewed are summarized in Table 1.

Table 1.

Expression and function of key ion channels in astrocytic endfeet, and ECs and SMCs of parenchymal arterioles.

	Astrocytic Endfeet	Notes, References	Parenchymal Endothelium	Notes, References	Parenchymal Smooth muscle	Notes, References
Membrane potential (mV)	−87	In vivo, cell soma.⁷	−35 to −40?	Not measured. Inferred from observations that EC-SMC Vm is nearly equivalent in hamster feed arteries.⁸	−35 to −40	In situ, 40 mm Hg.^9,10
Ca²⁺ channels
IP₃R	+	Ca²⁺ wave generation.¹¹	+?	Not directly tested. Mediates Ca²⁺ signaling in other arteries.¹²	+?	Not directly tested. UTP evokes Ca²⁺ waves in pial arteries.¹³
RyR	–	Not present.¹¹	–?	Not directly tested. Not present in other arterioles.	+	Ca²⁺ waves in situ at 40 mm Hg. Ca²⁺ sparks in response to H⁺.¹⁴
VDCC	–?	Nifedipine inhibition of Ca²⁺ waves.¹⁵	–?	Not directly tested. Not present in other arterioles.	+	Depolarization-evoked Ca²⁺ entry leading to constriction.⁹
TRPV4	+	Ca²⁺ entry and wave propagation.¹⁶	+?	Not directly tested. Present in pial endothelium.^17,18	+	Ca²⁺ influx.¹⁹
TRPC6/M4	n.d.		–	No effect of endothelium removal on TRPM4 responses.²⁰	+	TRPM4 contributes to myogenic tone.²⁰ Both C6 and M4 contribute to tone in pial arteries.²¹
K⁺ channels
BK	+	Vasoactive K⁺ release.^22,23	–	Not present in acutely isolated cells.¹⁰	+	Present in situ. Low basal activity at 40 mm Hg.¹⁰
SK	–	Not detected.²⁴	+	Present in acutely isolated cells.¹⁰	–	No effect of NS309 after endothelial removal.¹⁰
IK	+	Detected in processes and endfeet. Engaged in NVC.²⁴	+	Present in acutely isolated cells.¹⁰	–	No effect of NS309 after endothelial removal.¹⁰
Kv	–?	Not directly tested. Membrane depolarization sufficient to engage Kv is unlikely to occur.	–	No voltage-activated currents after SK/IK blockade.¹⁰	+	Tonically active in situ at 40 mm Hg. Resists constriction.²⁵
K_IR2	+?	Not directly observed in endfeet, but expressed by astrocytes.²⁶	+	Present in other arterioles.²⁷ Preliminary data indicates present in parenchymal endothelium.²⁶	+	Activated by extracellular K⁺.^22,23,28,29

Summary of the expression of ion channels involved in neurovascular coupling (NVC) in native preparations of astrocytic endfeet, parenchymal arteriole ECs and SMCs. Studies in culture systems were not considered. Question marks are used to highlight inferences drawn in cases where channels have not been directly observed in a particular cell type.

EC-SMC Vm: endothelial cell/smooth muscle cell membrane potential; VDCC: voltage-dependent calcium channel; TRP: Transient receptor potential; BK: large-conductance calcium-activated potassium channel; SK: small-conductance calcium-activated potassium channel; IK: intermediate-conductance calcium-activated potassium channel; Kv: Voltage-dependent potassium channel; n.d.: no data.

The neurovascular unit

The neurovascular unit (NVU) consists of neurons, astrocytes and the cells of parenchymal arterioles, which consist of a single layer of SM cells (SMCs) surrounding the endothelium (Figure 1a). Parenchymal arterioles originate from pial (surface) vessels and penetrate into the brain, where they become almost completely encased by astrocytic endfeet.^30,31 This positions endfeet to act as intermediates between neurons and the vasculature. Similarly, endothelial membrane extensions project through fenestrations in the internal elastic lamina and basement membrane of arterioles to directly contact SMCs (Figure 1b). These structures—termed myoendothelial projections (MEPs)—not only provide direct contact between these two cell types through gap junctions but also offer a unique intracellular and extracellular microdomain signaling environment for controlling vascular tone. In contrast, direct cell–cell contact has not been observed between astrocytic endfeet and SMCs, although the membranes of these cells are closely opposed. Each parenchymal arteriole supplies a large territory of downstream capillaries, an anatomical organization that positions parenchymal arterioles as bottlenecks to the entry of blood into the brain.³² Therefore, control of parenchymal arteriole diameter by NVC mechanisms is of vital importance to the regulation of downstream blood flow.

Figure 1.

Anatomical features of the NVU and MEPs. (a) Electron micrograph depicting astrocytic endfeet (EF) enveloping a parenchymal arteriole with a single layer of SMCs and underlying ECs. Adjacent to the endfeet is the brain parenchyma (P) containing neuronal and astrocytic processes. Scale bar: 10 µm. (b) A MEP site through a fenestration in the internal elastic lamina (IEL) between an EC and SMC in a human parenchymal arteriole. Black arrowheads indicate a myoendothelial gap junction. Scale bar: 250 nm. Reproduced with permission from Aydin et al.³³

The ionic composition in the NVU establishes the basal conditions for controlling cerebral blood flow

The choroid plexuses produce cerebrospinal fluid (CSF), which fills the ventricles and the subarachnoid space and circulates from the latter into the brain parenchyma via the Virchow-Robin space, taking a paravascular route through the ‘glymphatic’ system.³⁴ The composition of CSF is distinct from that of plasma (see Table 2, reproduced from Brown et al.³⁵). Thus, with the luminal surface of ECs exposed to plasma and the parenchymal surface of SMCs bathed in CSF, SMCs and ECs experience extracellular milieus with different ionic compositions. The concentrations of ions in these extracellular compartments dictate their equilibrium potentials and, by extension, both ion channel activity and V_m. This, in turn, influences the level of resting SM tone and tissue perfusion. Variations in local ion concentrations during neuronal activity modulate the SM V_m, thereby exerting a powerful effect on parenchymal arteriolar diameter; this is the central relationship underlying the dynamic regulation of cerebral blood flow.

Table 2.

The composition of plasma and cerebrospinal fluid.

	Plasma	CSF
Na⁺ (mM)	155	151
K⁺ (mM)	4.6	3.0
Mg²⁺ (mM)	0.7	1.0
Ca²⁺ (mM)	2.9	1.4
Cl⁻ (mM)	121	133
HCO₃⁻ (mM)	26.2	25.8
Glucose (mM)	6.3	4.2
Amino acids (mM)	2.3	0.8
pH	7.4	7.4
Osmolality (mosmol.Kg H₂O⁻¹)	300	305
Protein^a (mg 100 g⁻¹)	6500	25

Rabbit CSF; all other values are for dog CSF. Reproduced from Brown et al.,³⁵ with permission.

Part I: control of the smooth muscle membrane potential is central to the control of cerebral blood flow

The key role of the voltage-dependent Ca²⁺ channel as a membrane potential biosensor

The V_m of SM exerts profound control over cerebral artery diameter.^9,36 In isolated parenchymal arterioles, a graded increase in intravascular pressure, for example from 5 mm Hg to 40 mm Hg, causes graded V_m depolarization from approximately –60 mV to –40 mV, an elevation in intracellular calcium ([Ca²⁺]_i) from ∼120 nM to ∼250 nM, and approximately 50% constriction⁹ (Figure 2). In contrast, V_m hyperpolarization to –60 mV causes near-maximal vasodilation (i.e. almost equivalent to the passive diameter of the vessel).^9,36 The depolarization/vasoconstriction to increasing pressure is termed the ‘myogenic response’ and is an intrinsic property of small resistance arterioles from a range of vascular beds.³⁷ In brain arterioles, the myogenic response is particularly important for cerebral autoregulation, in which vessels constrict or relax to maintain a constant level of brain perfusion despite varying peripheral blood pressure.³⁰ Therefore, myogenic regulation sets the level of resting perfusion, while also providing a baseline tone from which arterioles can dilate or constrict in response to factors released during neuronal activity. As such, myogenic mechanisms can be viewed as the foundation upon which NVC mechanisms are constructed.

Figure 2.

Relationships between intravascular pressure, vessel diameter, [Ca²⁺]_i and V_m in pial arteries and parenchymal arterioles. (a) One fundamental difference between vessels of the cerebral circulation is that parenchymal arterioles develop more tone in response to lower intravascular pressure compared to pial arteries. (b) The phenomenon in A is linked to the parenchymal arteriole SM V_m, which is more depolarized in response to lower pressure compared to pial arteries. (c) Increases in [Ca²⁺]_i in response to increasing intravascular pressures are greater in parenchymal arterioles than in pial arteries owing to higher voltage-dependent calcium channel (VDCC) activity caused by the greater degree of SMC cell depolarization, as illustrated in (b). (d) There is no difference in the sensitivity of the SM contractile apparatus to Ca²⁺ between pial arteries and parenchymal arterioles, indicating that the difference in the pressure-constriction relationship between these two types of vessels is due to the difference in SM V_m in response to pressure. Data were re-plotted from ref.⁹ Parenchymal arteriole V_m data were obtained from F. Dabertrand (personal communication) and from Nystoriak et al.⁹ and Hannah et al.¹⁰

Pial arteries on the surface of the brain develop less myogenic tone in response to pressure compared with parenchymal arterioles, despite the fact that the relationship between SM [Ca²⁺]_i and constriction is identical in both of these types of vessel (see Figure 2d). This is because parenchymal arteriole SM is more depolarized than pial SM at lower pressures (Figure 2b) and thus exhibits a greater influx of Ca²⁺ (Figure 2c) and greater constriction (Figure 2a). The molecular arrangement that leads to the higher sensitivity of parenchymal arterioles to pressure has not yet been elucidated but likely involves a loss of negative feedback control of the SM V_m by Ca²⁺ spark-BK channel interactions (see ‘Ryanodine Receptors in Smooth Muscle’, below).

L-type VDCCs are the key voltage sensors in vascular SM.³⁸ These channels translate changes in the V_m into alterations in [Ca²⁺]_i and thereby adjust the contractile state of the cell. Their central importance is evidenced by the fact that V_m changes induced by pressure or other agents have no effect on vascular tone in the presence of VDCC inhibitors,³⁶ highlighting the vital role of Ca²⁺ entry through L-type VDCCs in vasoconstriction. Parenchymal arteriolar myocytes express L-type VDCCs composed of pore-forming Ca_V1.2 α_1C-subunits and associated α₂δ-, β- and γ-subunits.^9,39 Recent studies have also presented evidence for the expression and function of T-type VDCCs in SMCs of pial arteries.^40,41 However, no contribution of T-type channels to either [Ca²⁺]_i or arteriolar tone has been shown at physiological pressures in isolated parenchymal arterioles.⁹

Neurovascular signaling mediators that converge on the SM utilize a variety of mechanisms to drive changes in V_m. Critically, this leads to either (1) closure of VDCCs (in the case of membrane hyperpolarization), a fall in global [Ca²⁺]_i and vasodilation or (2) depolarization and an increase in VDCC open probability (Po), causing an increase in [Ca²⁺]_i and vasoconstriction.^38,42 Thus, tuning the SM V_m to modulate Ca²⁺ entry through VDCCs is the key mechanism for controlling the diameter of parenchymal arterioles and rapidly influencing cerebral blood flow.

A role for smooth muscle transient receptor potential channels in myogenic depolarization: evidence from pial and parenchymal arterioles

Transient receptor potential (TRP) channels are a diverse family of cation-permeable channels activated by a wide range of stimuli.⁴³ Members of the TRP family share an overall structural similarity, forming tetrameric homomeric or heteromeric channels from subunits containing six transmembrane domains. TRP channels are broadly classified on the basis of sequence homology into six subfamilies in mammals: TRPA (ankyrin), TRPC (classic or canonical), TRPM (melastatin), TRPML (mucoliptin), TRPP (polycystin) and TRPV (vanilloid).⁴⁴ Most of these channels act as routes for the entry of Ca²⁺, although some, such as a subset of TRPM channels, primarily mediate Na⁺ entry. For a comprehensive overview of TRP channels in the vasculature, see Earley and Brayden.⁴⁵

Mounting evidence suggests that intravascular pressure or vasoconstrictor agonists can cause V_m depolarization through the activation of TRP channels in pial artery myocytes; this, in turn, increases the activity of VDCCs and promotes vasoconstriction. TRPV4, TRPC3, TRPC6 and TRPM4 channels have all been identified in pial SM,^46–49 and recent studies indicate that pressure causes depolarization through activation of TRPM4 and TRPC6 channels. Monovalent cation-permeable TRPM4 channels, though impermeable to Ca²⁺ ions,⁴⁴ are activated by sarcoplasmic reticulum (SR) 1,4,5-trisphosphate receptor (IP₃R)-mediated Ca²⁺ release events in pial artery myocytes.⁵⁰ This results in an influx of Na⁺ through TRPM4, which can be measured as a transient inward cation current (TICC).⁵⁰ Pressure-induced constriction of ex vivo cerebral arteries requires functional TRPM4 channels,⁴⁶ and blocking TRPM4 causes almost maximal vasodilation of pressurized arteries,⁵¹ suggesting that these channels are vitally important for the myogenic response. Similarly, activation of TRPC6 channels, which have a Ca²⁺:Na⁺ permeability ratio of ∼6:1,⁵² is another contributor to pressure-induced constriction of isolated pial arteries, as evidenced by the fact that knockdown of TRPC6 using antisense oligonucleotides attenuates the myogenic response.⁴⁷ A recent study,²¹ conducted in isolated myocytes and pressurized pial arteries, unifies the contributions of TRPC6 and TRPM4 into a single mechanism for myogenic constriction. Here, pressure-induced activation of phospholipase C (PLC) γ1 liberates IP₃ and diacyl glycerol (DAG). Subsequently, DAG activates TRPC6 channels directly and the resulting Ca²⁺ influx across the sarcolemma acts synergistically with IP₃ to activate IP₃Rs, releasing Ca²⁺ from the SR that then activates TRPM4 channels to depolarize the membrane.²¹

Despite these major advances in our understanding, the mechanism by which pressure activates TRP channels has not been firmly established. In addition to the possibility that TRP channels directly sense membrane deformation,⁵³ they may be attached to other cellular structures that respond to pressure, such as the cytoskeleton.⁵⁴ Alternatively, upstream mechanosensitive signaling elements may transduce physical forces into second messenger signaling (such as PLC activation),²¹ which is then detected by TRP channels.⁵⁵ These possibilities, which are not mutually exclusive, have recently been reviewed in detail.⁵⁶

In contrast to the vasoconstriction associated with TRPC6 and TRPM4 activation, SM TRPV4 channels, which are non-selective but possess a high permeability to Ca²⁺, have been implicated in vasodilatory mechanisms in pial arteries.⁴⁹ According to this mechanism, reported by Earley et al., epoxyeicosatrienoic acids (EETs) activate TRPV4 channels, which conduct Ca²⁺ into the cell and enhance ryanodine receptor (RyR)-mediated Ca²⁺ spark activity (see below),⁴⁹ resulting in vasodilation.⁵⁷

The studies described above were conducted using pial arteries. Recently, Li et al.²⁰ pioneered the exploration of SM TRP channel contributions to myogenic tone in parenchymal arterioles. In keeping with their myogenic role in pial arteries, TRPM4 channels were found to be vitally important for the development of tone in isolated arterioles from this vascular bed. Here, TRPM4 activation and membrane depolarization lies downstream of direct mechanoactivation of purinergic P2Y₄ and P2Y₆ G_q-type G-protein coupled receptors (G_qPCRs) and consequent stimulation of PLC activity by intravascular pressure,^20,58,59 suggesting that mechanisms similar to those reported for pial arteries may also be at play in parenchymal arterioles (Figure 3).

Figure 3.

The central roles of SM TRPM4 channels and VDCCs in myogenic constriction of parenchymal arterioles. Intravascular pressure (∼40 mm Hg⁶⁰) activates G_q-coupled P2Y receptors on the SM, leading to PLC activation. Through an as-yet-undefined pathway in parenchymal arterioles (see text for insights from pial arteries²¹), PLC activation leads to a depolarizing Na⁺ influx through TRPM4, triggering Ca²⁺ influx through VDCCs and leading to myocyte contraction.²⁰

Ryanodine receptors in smooth muscle: Ca²⁺-release channels that oppose myogenic constriction

Although extracellular Ca²⁺ entry through VDCCs has a central role in the delivery of Ca²⁺ for SM contraction, Ca²⁺ release from the SR through RyRs has the opposite effect.⁶¹ Thus, Ca²⁺ in SM has a dichotomous role, with distinct Ca²⁺ signaling modalities either promoting or opposing vasoconstriction.

RyRs are homotetrameric assemblies located on the sarco-/endoplasmic reticulum (SR/ER) in many cell types. They are gated by Ca²⁺ and modulated by Mg²⁺, ATP, calmodulin and FK506-binding proteins as well as by phosphorylation by protein kinases such as protein kinase A (PKA), Ca²⁺/calmodulin-dependent kinase II and cGMP-dependent protein kinase (PKG).⁶² Ca²⁺ release through RyRs can lead to two types of signal: waves and sparks. Ca²⁺ waves are propagating events with considerable spatial spread that result from Ca²⁺-induced Ca²⁺ release (CICR), a process whereby the presence of a sufficiently high concentration of local Ca²⁺ evokes Ca²⁺ release from nearby RyRs and/or IP₃Rs in the SR membrane. This released Ca²⁺ then evokes further release from adjacent channels; the process repeats, leading to a regenerative Ca²⁺ wave that propagates along the SR. The role of Ca²⁺ waves in vascular SM is not fully understood, although waves in pial arteries may contribute to myogenic tone development, particularly at lower pressures.⁶³ In contrast, Ca²⁺ sparks are highly localized, brief events mediated by the simultaneous opening of several clustered RyRs that cause local Ca²⁺ to reach micromolar concentrations.^61,64,65 In pial artery SM, sparks occur at physiological pressures (60 mm Hg).⁶⁶ In these vessels, and a range of other vascular beds, large-conductance Ca²⁺-activated K⁺ (BK) channels are localized in the sarcolemma adjacent to SR RyR Ca²⁺ spark sites. These channels are activated by sparks, releasing K⁺ and resulting in a spontaneous transient outward current (STOC) that causes a brief hyperpolarization.⁶¹ In the pial circulation, this process acts as a negative feedback on membrane depolarization and Ca²⁺ entry through VDCCs^61,67 and thus is an important determinant of arteriolar tone.

In contrast to pressurized pial arteries, where Ca²⁺ spark activity is a prominent feature, spark activity is absent in the SM of ex vivo pressurized (40 mm Hg) parenchymal arterioles; instead, asynchronous ryanodine-sensitive Ca²⁺ waves are the predominant form of Ca²⁺ signal here.¹⁴ However, the RyR Ca²⁺ spark-BK channel STOC signaling architecture is still present and can be engaged under certain circumstances,¹⁴ making this a potential target for neurovascular signaling mediators. Dabertrand and co-workers¹⁴ demonstrated this principle by acidifying artificial CSF from pH 7.4 to pH 7.0, which converted SM RyR-sensitive Ca²⁺ waves to sparks in parenchymal arterioles. The resultant increase in spark activity opposed pressure-induced vasoconstriction and led to a rapid and sustained vasodilation that was sensitive to both ryanodine and the BK channel blocker paxilline in a non-additive manner.¹⁴ It is presently unclear why Ca²⁺ sparks do not occur under basal (experimental) conditions in isolated parenchymal arterioles, but this observation may help to explain why these arterioles develop higher tone at lower pressures compared with pial arteries. Indeed, one possibility is that RyRs in parenchymal arterioles have a higher basal open probability than their counterparts in pial arteries.⁶⁸ Such higher basal sensitivity to Ca²⁺ could lead to the preferential generation of waves rather than sparks in parenchymal arterioles, leaving an absence of the hyperpolarizing influence of spark-STOC signaling and higher tone. This contrasts with pial arteries, where basal spark-STOC signaling activity acts as a negative feedback that resists SM membrane depolarization, resulting in lower tone at the same pressure.

IP₃ receptors

IP₃Rs are ubiquitous ligand-gated Ca²⁺-permeable channels located in the membrane of the SR/ER.⁶⁹ To date, IP₃R signaling in parenchymal arteriole SM has not been examined, but in pial arteries, UTP-induced PLC activation has been linked to IP₃R-dependent Ca²⁺ waves that lead to vasoconstriction.¹³ Moreover, Xi and co-workers identified a possible physical interaction of IP₃Rs with TRPC3 in pial SM that was independent of SR Ca²⁺ release. The resultant cation current was demonstrated to promote vasoconstriction in response to endothelin-1.⁴⁸ Since TRPC3 channels do not participate in pressure-induced constriction,⁷⁰ this interaction might be important for receptor activation-induced constriction of cerebral arteries. As previously noted, pial SM IP₃R activation leads to depolarizing TRPM4 currents.²¹ Since TRPM4 channels have a similar role in parenchymal arterioles,²⁰ IP₃R-mediated Ca²⁺ signaling is a likely candidate for mediating activation of TRPM4 channels here as well. IP₃Rs are also involved in activation of the Ca²⁺-dependent transcription factor NFATc3 and subsequent regulation of pial SM gene expression.⁷¹

K⁺ channels in smooth muscle

The resting V_m of SM (−35 to −45 mV) indicates that the ratio of K⁺ permeability (pK) to the permeability of other ionic species (assuming a collective reversal potential of 0 mV) is around 0.5–0.8:1.²⁶ Thus, the SM V_m is quite positive to the K⁺ equilibrium potential (E_K), which is approximately −103 mV in CSF (assuming 3 mM extracellular K⁺ and 140 mM intracellular K⁺). These basal conditions mean that the activation of K⁺ channels can impart a major hyperpolarizing influence on cerebral SM, and thereby exert rapid and robust control over vascular diameter.

Smooth muscle inwardly rectifying K⁺ channels: Targets of endfoot K⁺ release

A modest elevation of external K⁺ is a rapid and powerful vasodilator of pial arteries and parenchymal arterioles.^22,23 For example, raising K⁺ from 3 mM to 8 mM hyperpolarizes the SM in isolated pressurized parenchymal arterioles to near the new E_K of −76 mV, and causes maximum vasodilation.²² For this to occur, K⁺ permeability has to increase more than 50-fold; this is achieved through activation of SM inwardly rectifying K⁺ (K_IR) channels.²⁶

K_IR channels, which form tetramers composed of two-transmembrane α-subunits, can be distinguished by their rectification properties.⁷² The K_IR subtypes expressed in parenchymal arterioles have yet to be definitively established, but the pharmacological sensitivity of the parenchymal SM K_IR current to Ba²⁺ ions in situ^22,28 is consistent with the presence of functional K_IR2-containing channels. In addition, mRNA data indicate the expression of K_IR2.1 and K_IR2.2 isoforms in rat parenchymal arterioles.²⁸ In pial arteries, knockout of K_IR2.1, but not K_IR2.2, completely ablates the dilatory response of vessels to small increases in extracellular K⁺,²⁹ suggesting a principal role for the K_IR2.1 subtype. Furthermore, downregulation of SM K_IR2.1 expression leads to impairment of K⁺-mediated dilation of rat parenchymal arterioles,²⁸ strongly suggesting that K_IR2.1 is of similar central importance in these smaller vessels. K_IR2 family members are strongly rectifying and are activated by hyperpolarization and external K⁺, with their half-activation voltage corresponding to E_K.⁷³ At potentials positive to E_K, outward K⁺ current through K_IR channels progressively declines with depolarization as a result of intracellular polyamine block.⁷⁴ As described previously, at basal extracellular K⁺ (3 mM), E_K is approximately −103 mV and the SM V_m is about −35 to −40 mV.^9,10 Under these conditions, K_IR channel activity is very low.²⁶ K⁺ is released from neurons and from endfoot BK channels during NVC (see below), and raising K⁺ by a small amount (e.g., to 8 mM) leads to rapid unblock of K_IR channels and an enormous increase in K_IR conductance.²⁶ The ensuing increase in K⁺ permeability drives the SM V_m to the new E_K (−76 mV with 8 mM [K⁺]_o), in the process promoting even greater K_IR channel activation by virtue of their steep voltage dependence.²⁶ This ∼40 mV hyperpolarization closes VDCCs and leads to maximal vasodilation, making K_IR activation by K⁺ released during neuronal activity a powerful mechanism for increasing local cerebral blood flow. The stark hyperpolarizing effect of K⁺ on parenchymal SM V_m likely also involves K_IR channels located on the underlying endothelium, for which there exists preliminary evidence.²⁶ Here, K⁺ may diffuse through the SM, or hyperpolarizing signals could be conducted upstream from capillaries (see below), to activate EC K_IR channels. This would promote EC hyperpolarization which can be transmitted via gap junctions to reinforce SM hyperpolarization, further amplifying K_IR channel activation and locking V_m at E_K (Figure 4). For an extended discussion of K⁺-induced activation of parenchymal arteriole K_IR channels in the control of cerebral blood flow, see Longden et al.²⁶

Figure 4.

Feed-forward hyperpolarization of SMCs by K_IR channel activation by K⁺ and V_m. A small amount of K⁺ released from neurons or astrocytes during neuronal activity may activate K_IR channels on the SM and possibly also diffuse to K_IR channels on ECs. Activation of K_IR channels initiates membrane hyperpolarization, in the process promoting further K_IR channel activation to amplify hyperpolarization and lead to V_m reaching E_K. EC hyperpolarization, either due to K⁺ activation of K_IR or resulting from conducted signaling from downstream capillaries, could also be transmitted to the SMs by gap junctions, further amplifying SMC hyperpolarization.

K_IR subunits are also components of ATP-sensitive K⁺ (K_ATP) channels, which are found in many peripheral vascular beds. K_ATP channels, composed of K_IR6.1 or 6.2 with one of four types of auxiliary sulfonylurea (SUR) receptors, are weakly rectifying and their gating is determined by local concentrations of intracellular nucleotides.⁷² K_ATP channels composed of K_IR6.1 and K_IR6.2 plus SUR2B subunits are expressed in basilar and middle cerebral pial arteries⁷⁵ and may play a role in the dilatory responses of large surface arterioles to hypoxia⁷⁶ and acidosis.⁷⁷ However, these channels appear to be absent from the SM of parenchymal arterioles, as evidenced by a lack of vasodilatory response to the K_ATP channel opener cromakalim.¹⁴

Ca²⁺-activated K⁺ channels: the hyperpolarizing influence of smooth muscle BK channel activity can be exploited by NVC mediators

The Ca²⁺-activated potassium (K_Ca) channel family is divided into three subgroups: small-conductance (SK) channels consisting of K_Ca2.1 (SK1), 2.2 (SK2) and 2.3 (SK3) isoforms, the intermediate-conductance (K_Ca3.1; IK) channel and the large conductance (K_Ca1.1; BK) channel.⁷⁸ BK channel α-subunits possess six transmembrane domains with an extended intracellular C-terminus. These subunits gain their Ca²⁺-sensitivity from C-terminal regions, termed the Ca²⁺-bowl and the RCK (regulator of conductance for K⁺) domain. Four α-subunits associate with four β-subunits, the latter of which modulate the overall gating properties of the channel. α-subunits also possess a functional voltage sensor in their S4 region whose sensitivity is modified by local Ca²⁺ as well as by phosphorylation by a number of intracellular enzymes, making these channels responsive to a diverse range of intracellular stimuli.⁷⁹

In line with observations in other vascular beds, parenchymal myocytes express functional BK channels, but do not express SK or IK channels.¹⁰ In contrast to their important role in opposing pressure-induced constriction in pial artery SM,⁶⁷ BK channels contribute only modestly to parenchymal arteriolar tone.¹⁰ However, these channels represent a key target for many putative NVC mediators (e.g. EETs, nitric oxide and prostaglandins), which may harness BK channel activity to bring about membrane hyperpolarization.

K_V channels exert a tonic hyperpolarizing influence on the smooth muscle membrane potential

Voltage-dependent potassium (K_V) channels constitute the largest subfamily of the K⁺ channel superfamily.⁸⁰ Functional channels are formed from four six-transmembrane α-subunits, either homo- or heteromeric, plus additional β-subunits.⁸¹ Rat parenchymal arterioles express mRNA for K_V1.2 and K_V1.5 α-subunits.²⁵ Inhibiting K_V channels in parenchymal arterioles causes a significant constriction, indicating that these channels provide a hyperpolarizing K⁺ efflux that opposes vasoconstriction at physiological pressures.²⁵ Similarly, pial arteries express protein for K_V1.2 and K_V1.5 subtypes, which are thought to assemble into heterotetramers; these channels are tonically active in isolated pressurized pial arteries.⁸² Figure 5 provides an overview of the ion channel influences on SM V_m, the balance of which determines arteriolar tone at any given point.

Figure 5.

Schematic illustration of the central role of SM V_m in cerebrovascular constriction. Arrowheads indicate a stimulatory effect, whereas flat lines indicate a negative influence. TRP channel activity is engaged by pressure, which depolarizes the membrane and increases VDCC activity, leading to an increase in [Ca²⁺]_i and constriction. Membrane depolarization directly stimulates K_V channels, which mediate hyperpolarizing currents that, in turn, exert a negative feedback on depolarization; smooth muscle K_IR channel activation also counteracts depolarization. Under certain conditions, elevated [Ca²⁺]_i can induce RyR-mediated Ca²⁺ sparks, which couple to BK channels to hyperpolarize the membrane and limit depolarization. Astrocytic endfoot and endothelial signaling acts to hyperpolarize the SM membrane. The balance of these signaling elements controls Ca²⁺ entry into the myocyte and thus the contractile state of the cell. Adapted from Dabertrand et al.⁸³

Part II: dual control of arteriolar smooth muscle by the endothelium and astrocytic endfeet

Both the endothelium and astrocytic endfeet share a number of common features that allow them to control V_m and the contractile state of the SM sandwiched between them. Each cell type exhibits local modulated Ca²⁺ signals, which lead to the release of a similar complement of vasoactive substances onto the SM. In keeping with this theme, astrocytes and ECs also express a highly similar repertoire of ion channel species.

The astrocyte cell membrane possesses a large passive K⁺ conductance and a negative resting potential, typically measured in the −70 to −80 mV range in brain slice electrophysiological recordings.^84–86 One recent study using intracellular microelectrodes to record at the level of the astrocytic soma in vivo determined that the resting V_m was approximately −87 mV.^7,8 Although parenchymal endothelial V_m has yet to be directly measured, dual microelectrode recordings from ECs and SM in hamster feed arteries showed that the V_m of these two cell types were very nearly equivalent.⁸ Moreover, measurements performed on peripheral arterioles using a range of preparations and techniques^8,12,87–89 have established that endothelial resting V_m is between approximately −30 and – 45 mV. Thus, it is likely that parenchymal endothelial resting V_m lies within this range, closely matching that of the electrically connected overlying SM.

IP₃Rs and TRPV4 play central roles in Ca²⁺ signaling in the endothelium and endfoot

In contrast to the RyR-mediated Ca²⁺ signaling that predominates in SM, Ca²⁺ release through IP₃Rs has a key role in communication to the SM by both the parenchymal endothelium and astrocytic endfeet. Although the mechanisms by which extracellular Ca²⁺ ions enter the endothelium and endfoot remain unsettled, it is unlikely that VDCCs contribute to Ca²⁺ influx in either cell type. Instead, local Ca²⁺ entry through TRPV4 channels has been shown to be important in both ECs⁹⁰ and astrocytes.¹⁶

IP₃Rs

Astrocytes are equipped with a range of neurotransmitter receptors,⁹¹ enabling them to respond to a diverse array of neuronal signaling molecules. Glutamate is the predominant excitatory neurotransmitter in the brain, and the prevailing view is that astrocytes detect glutamatergic neuronal activity through G_q-coupled metabotropic glutamate receptors (mGluRs) expressed on processes that contact synapses.⁹² According to this model, activation of these G_qPCRs causes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) by PLC, resulting in the production of IP₃ and DAG.⁹³ IP₃, in turn, acts on IP₃Rs to increase their sensitivity to [Ca²⁺]_i and increase Ca²⁺ efflux from the ER (Figure 6). The IP₃R2 isoform is thought to be primarily responsible for the release of Ca²⁺ from astrocytic intracellular stores in hippocampal and cortical astrocytes, as some studies have observed that ablation of the gene encoding this channel attenuates G_qPCR-stimulated and neuronal activity-evoked astrocytic Ca²⁺ signaling.^94–96 Further studies, using IP₃R2-knockout mice, have raised questions about the role of IP₃R-mediated astrocytic Ca²⁺ signaling in the control of cerebral blood flow, reporting that functional hyperemia is unaffected in these mice.^97,98 While these studies raise the intriguing possibility of IP₃R2-independent NVC, their broader conclusion—that astrocytic Ca²⁺ signaling is dispensable for NVC and functional hyperemia—is premature and directly contradicts a substantial body of evidence to the contrary. For example, several laboratories have firmly established that uncaging Ca²⁺ or IP₃ in astrocytic endfeet leads to diameter changes in the adjacent arteriole in brain slices and in vivo in both rats and mice.^{23,11,99–101} It should also be noted that retention of NVC upon IP₃R2 knockout is not a universal finding⁹⁵; a recent study of IP₃R2-knockout mice using brain slices and in vivo preparations identified a diverse range of persistent astrocytic Ca²⁺ signaling events, localized mainly to fine processes and endfeet rather than somata.¹⁰² Further recent studies have confirmed that astrocytic, fast Ca²⁺ transients that occur in the cell soma, fine processes, and endfeet reliably precede functional hyperemia in adult mice in vivo.^103,104 Thus, it appears that astrocytes from IP₃R2-knockout mice do continue to engage in Ca²⁺ signaling, perhaps due to compensatory upregulation of other IP₃R isoforms, likely on a spatial and temporal scale that previous studies were unable to detect.

Figure 6.

Neurovascular communication is initiated by neuronal activity, which drives the production of IP₃ by PLC, in turn initiating a propagating Ca²⁺ wave. Ca²⁺ waves arriving at the endfoot activate TRPV4 channels in a positive-feedback loop that further enhances their spread, ultimately leading to the stimulation of Ca²⁺-sensitive ion channels and enzymes.

The astrocytic ER extends into the endfoot, as evidenced by the fact that both electrical field stimulation and direct photolysis of caged-IP₃ evoke Ca²⁺ release in endfoot processes adjacent to arterioles.¹¹ Thus, it is thought that the presence of sufficiently high concentrations of IP₃ generated in response to neuronal activity evoke regenerative CICR, leading to a Ca²⁺ wave that propagates into the astrocytic endfoot.¹¹ Arriving Ca²⁺ waves stimulate the production and/or release of numerous vasoactive molecules, which cause vasodilation and a resultant increase in local cerebral blood flow. Consistent with this, astrocytes in brain slices respond to electrical and pharmacological mGluR stimulation with an elevation in Ca²⁺.^11,105–107 These findings, which were obtained in juvenile animals, are compatible with an interesting recent study reporting that expression of G_q-coupled mGluRs is prominent in cortical and hippocampal astrocytes of young mice but is lost in adulthood.¹⁰⁷ However, the implication of this latter study—that expression of these receptors is a transient, developmental phenomenon—must be weighed against functional evidence that mGluR-mediated Ca²⁺ signaling in astrocytes is known to occur in adult animals in vivo.^99,108 These seemingly irreconcilable observations suggest that the signaling cascade leading to astrocytic Ca²⁺ waves may be more complex than currently conceived—particularly in mature animals¹⁰⁷—and indicate that further work is needed to fully understand this phenomenon.

Given that the overlying SM acts as a barrier to immediate neuronal or astrocytic communication, it is unlikely that parenchymal arteriolar endothelium is directly stimulated during NVC. However, it is plausible that conducted signaling from capillary ECs (see below) could lead to parenchymal arteriole EC engagement.¹⁰⁹ In ECs of small arteries of the periphery, Ca²⁺ release through IP₃Rs is well-established as an important signaling step leading to vasodilation. In particular, brief, high-amplitude, IP₃R-mediated Ca²⁺ signals localized specifically to MEP microdomains—termed Ca²⁺ pulsars—activate local IK channels, causing K⁺ efflux and hyperpolarization.¹² This hyperpolarization can then be transmitted via gap junctions directly to the underlying SM; the released K⁺ may also amplify hyperpolarization by activating local K_IR channels and/or Na⁺/K⁺ ATPase pumps on the endothelium and/or SM¹¹⁰ (Figure 7). Although currently unexplored, it is highly likely that this same signaling modality exists in parenchymal arteriole ECs, and if so, this could conceivably be engaged by conducted or blood-borne signals to further sculpt blood flow responses.

Figure 7.

Astrocytic endfoot and EC K_Ca channel influences on SM V_m. Astrocytic Ca²⁺ waves initiated by neuronal activity can activate BK and IK channels directly on the endfoot. K⁺ accumulates in the restricted extracellular space, activating SM K_IR channels. Subsequent K⁺ efflux through these channels hyperpolarizes the SM membrane, which decreases VDCC open probability and thereby lowers global [Ca²⁺]_i to promote vasorelaxation. On the luminal side of the SM, the endothelium may be engaged during neurovascular coupling (for example by conducted signaling from the capillary bed or by luminal factors such as altered shear), leading to an increase in EC Ca²⁺ that could engage IK and/or SK channels in MEP microdomains to drive EC membrane hyperpolarization and also raise local K⁺. EC membrane hyperpolarization could then be transmitted via gap junctions to the overlying SM. EC and SM K_IR channels and Na⁺/K⁺ ATPase pumps could also respond to released K⁺ to amplify hyperpolarization.

TRPV4 channels

In addition to the well-studied IP₃-mediated waves described above, recent evidence indicates that spatially restricted astrocytic Ca²⁺ signals are also vital for the astrocyte to carry out its diverse roles, including the control of cerebral blood flow. Several studies have identified TRPV4 channels in astrocytes.^16,111 TRPV4 channels are enriched in astrocytic processes and endfeet adjacent to blood vessels and the glia limitans.¹¹¹ Dunn et al.¹⁶ recently studied the contribution of TRPV4 signaling to endfoot Ca²⁺ handling during NVC. This study identified spatially restricted TRPV4-mediated Ca²⁺-entry events that couple to endfoot IP₃Rs and then propagate throughout the endfoot as a Ca²⁺ wave¹⁶ (Figure 6). Inhibition of TRPV4 prior to neuronal stimulation was shown to substantially reduce the magnitude of the evoked endfoot Ca²⁺ transient, suggesting that TRPV4 is an important contributor to endfoot Ca²⁺ entry during neurovascular signaling. Although these channels can be activated by EETs, Dunn and co-workers found no evidence for EET-mediated activation of TRPV4 during neuronal activity. Instead, the authors proposed that IP₃R-mediated Ca²⁺ release engages Ca²⁺-sensitive TRPV4 channels in the endfoot membrane, stimulating further Ca²⁺ entry into the cytosol to help propagate the wave. These data also raise the intriguing possibility of TRPV4 activation by local physical forces.^55,112 For example, mechanical activation of TRPV4 by diameter changes in blood vessels could lead to endfoot Ca²⁺ increases that then either feedback to modulate vessel diameter and blood flow¹¹³ or propagate in retrograde fashion towards the astrocyte soma, where they might influence signaling to neurons.^16,114

In ECs of mesenteric arteries, Ca²⁺-entry events mediated by the gating of individual TRPV4 channels—termed ‘TRPV4 sparklets’—act as powerful regulators of vascular tone.⁹⁰ Here, endothelial Ca²⁺ entry though TRPV4 evoked by synthetic agonists primarily activates IK channels leading to vasodilation.⁹⁰ In the cerebral circulation, TRPV4 channels have been identified in the endothelium of the isolated middle cerebral (pial) arteries, where they contribute to Ca²⁺ entry in response to stimulation of purinergic G_qPCRs with UTP.¹⁷ Similarly, Hamel and co-workers¹⁸ recently confirmed the presence of functional TRPV4 channels in the endothelium of pressurized posterior cerebral (pial) arteries, showing that these channels link muscarinic receptor stimulation (with acetylcholine) to IK and SK channel activation. In the mesenteric endothelium, TRPV4 is co-localized with other signaling molecules at MEP sites,⁹⁰ where cooperative gating among TRPV4 channels in a four-channel cluster is enhanced by localized concentrations of A-kinase anchoring protein (AKAP) 150.¹¹⁵ Similarly, TRPV4 is also found at MEPs in cremaster arterioles.¹¹⁶ This same clustered organization may also occur at MEPs in cerebral arteries/arterioles, a hypothesis supported by preliminary evidence.¹¹⁷ To date, TRPV4 expression and function have not been explored in the parenchymal endothelium, but given its importance in mesenteric and pial ECs, this channel is a likely candidate for mediating Ca²⁺ entry into ECs in parenchymal arterioles as well.

Expression of Ca²⁺ targets in astrocytic endfeet and the endothelium

K_Ca channels and K⁺ signaling: a mechanism for bi-directional control of parenchymal arteriole diameter

Among the key targets of endfoot Ca²⁺ increases are K_Ca channels. There is good evidence for the functional expression of BK channels in the endfoot membrane adjacent to underlying parenchymal arterioles.^22,118 Astrocytes also express IK channels on processes and endfeet, whereas evidence for the functional expression of SK channels in astrocytes is currently lacking.²⁴ During neurovascular communication, IP₃-mediated Ca²⁺ waves arriving at the endfoot stimulate the opening of BK,^22,23 and possibly IK,²⁴ channels. K⁺ ions then accumulate in the extracellular nano-space between the endfoot and SM.^22,23 This increase in local K⁺ concentration induces hyperpolarization and relaxation of the SM through activation of myocyte (and possibly endothelial²⁶) K_IR channels²² (Figure 7). In addition to being supported by in situ and in vivo experimental data,^22,23 a number of the features of this mechanism have been confirmed using computational modeling.^119–111

The magnitude of the neuronally evoked endfoot Ca²⁺ increase dictates relative BK channel activity and thus the concentration of K⁺ released into the nano-space between endfeet and arterioles. Notably, K⁺ can promote vasodilation or vasoconstriction depending on its concentration, inducing dilation at <20 mM through activation of K_IR channels, and constriction at higher concentrations through depolarization of the SM and activation of VDCCs.²³ Accordingly, the degree of Ca²⁺ elevation in the astrocytic endfoot is a vital determinant of the polarity of the vascular response. This was demonstrated in experiments showing that endfoot Ca²⁺ levels could be manipulated by varying the intensity of electrical field stimulation or by carefully controlling uncaging of Ca²⁺ directly in the endfoot. Ca²⁺ levels less than ∼500 nM consistently caused dilation, whereas higher concentrations induced constriction. Both dilations and constrictions were sensitive to block by paxilline, supporting a model in which K⁺ release through endfoot BK channels mediates both vasodilation and vasoconstriction, depending on the intensity of the stimulus.²⁴

In contrast to astrocytic endfeet, the parenchymal endothelium expresses IK and SK channels, which contribute to basal parenchymal arteriole tone.¹⁰ SK and IK channels share a common topology, consisting of homomeric tetramers composed of four six-transmembrane α-subunits associated with intracellular Ca²⁺-binding calmodulin subunits at their C-terminal domains.⁷⁹ This renders these channels exquisitely sensitive to changes in [Ca²⁺]_i.¹²² Their vestigial voltage sensor, which contains fewer charged amino acids in the S4 transmembrane domain than that of K_V channels, means that SK and IK channels are voltage-insensitive.¹²³ Hannah et al.¹⁰ reported that blocking IK and SK channels in isolated pressurized parenchymal arterioles caused substantial vasoconstriction, and this same manipulation decreased resting cortical cerebral blood flow by 15% in vivo. Conversely, activation of these channels with NS309 maximally dilated isolated arterioles and greatly enhanced cortical cerebral blood flow, highlighting the role of these channels as powerful controllers of parenchymal arteriolar tone.¹⁰ In contrast, isolated, pressurized middle cerebral arteries do not constrict to IK and SK channel inhibition, suggesting that these channels are not tonically active under basal conditions in larger surface arteries.¹²⁴ In mesenteric arteries, the SK3 subtype has been identified at MEPs and EC-EC borders.¹²⁵ The loss of apamin-sensitive K_Ca currents in mesenteric ECs from an SK3 gene suppression mouse confirms that this is the primary SK isoform for the regulation of vascular tone.¹²⁶ SK2 is also present in ECs, but appears to be restricted to peri-nuclear regions of the cell, whereas SK1 is not present.¹²⁷

Engagement of SK and IK channels is dependent on [Ca²⁺]_i; thus, the above observations suggest a level of tonic endothelial Ca²⁺ signaling in parenchymal arterioles in situ and in vivo. Extrapolating from mesenteric arteries, where IP₃-mediated Ca²⁺ pulsars activate IK channels within MEPs¹² and thereby contribute to the control of vascular tone, it is possible to suggest that this same signaling architecture is present in the parenchymal circulation. If further studies establish that this is the case, K⁺ released through IK and SK channel activation by Ca²⁺ pulsars would be predicted to couple to local K_IR channel and Na⁺/K⁺ ATPase activation, which could act as an amplification mechanism to hyperpolarize the membrane and drive vasorelaxation¹¹⁰ (Figure 7).

Arachidonic acid metabolites

One early proposal¹²⁸ was that neuronally evoked Ca²⁺ waves activate Ca²⁺-sensitive cytosolic phospholipase A₂ in astrocytes, mobilizing arachidonic acid (AA) from membrane phospholipid pools and leading to subsequent metabolism of AA to EETs by cytochrome P450 2C11 enzymes. The four possible EET regioisomers—5,6-, 8,9-, 11,12- and 14,15-EET—act as short-range signaling hormones, dilating cerebral arteries in situ¹²⁹ and contributing to the control of cerebral blood flow in vivo.¹³⁰

EETs are synthesized by astrocytes in culture,¹³¹ and cytochrome P450 2C11 mRNA has been detected in perivascular astrocytes.¹³⁰ Astrocyte-derived EETs could potentially act in either an autocrine or paracrine manner to open endfoot¹³² or SM¹³³ BK channels, respectively. In the case of endfoot BK channels, this would cause the release of K⁺, which could then stimulate SM K_IR channel activity.¹³² If EETs diffuse to the SM, they could open BK channels to hyperpolarize the membrane directly.¹³³ The mechanism of EET action at BK channels involves ADP-ribosylation of G_sα subunits,¹³⁴ resulting in an increase in channel activity.¹³⁴ Astrocyte-derived EETs could also promote vasodilation through actions on TRPV4 channels in the endothelium,^17,18 myocytes,^19,49 and/or endfeet.¹⁶ Despite these possibilities, recent in vivo data argue against a direct contribution of endfoot-derived EETs to SM relaxation during NVC, as vasodilation evoked by uncaging Ca²⁺ directly in astrocytic endfeet in vivo was insensitive to cytochrome P450 blockade.⁹⁹ These observations suggest that the mechanism underlying the contribution of astrocyte-derived EETs to NVC is more complex and indirect than was first envisioned.

AA liberated by PLA₂ can also serve as a substrate for the production of vasoactive prostaglandins (PGs). In this reaction, the dual peroxidase-cyclooxygenase actions of cyclooxygenase (COX) enzymes initially metabolize AA to PGG₂ followed by the rapid conversion of this intermediate to PGH₂. PGH₂ is metabolized by cytochrome P450 enzymes to a range of PG products. Of these, PGE₂ has been proposed to play a role in NVC.¹⁰⁵ In this model, PGE₂ is liberated from the endfoot to act upon E-prostanoid (EP) receptors on SMCs. Notably, EP₄ receptors are G_s-coupled GPCRs, and stimulation of these receptors leads to activation of PKA, which can produce vasodilation.¹³⁵ However, recent studies have raised questions about the astrocytic involvement in the vasodilatory response to COX products. One group reported that COX-1 and -2 are expressed in astrocytes in juvenile rat hippocampal-neocortical slices and showed that pharmacological blockade of COX activity inhibited in situ vasodilations evoked by mGluR agonists and astrocyte Ca²⁺ uncaging.¹³⁶ However, another group found that COX-1 mRNA was expressed in only 10% of rat cortical astrocytes, whereas COX-2 was absent.¹³⁷ This same group recently identified a sub-population of COX-2 expressing pyramidal neurons as the primary cell type able to synthesize PGE₂, which reportedly acts through G_s-coupled EP₂ and EP₄ receptors to produce hyperemia.^137,138 However, a fundamental issue is raised by a recent study showing that application of PGE₂ directly to isolated, pressurized parenchymal arterioles from both rat and mouse evoked constriction rather than dilation, through activation of EP₁ receptors, suggesting that PGE₂ is unlikely to act as a direct mediator of NVC on parenchymal arteriole smooth muscle.¹³⁹ However, this does not rule out PGE₂-mediated vasodilatory effects through indirect actions on neurons¹³⁹ or on the capillary endothelium.

Liberation of AA from plasma membrane phospholipids by PLA₂ also occurs in the endothelium. In contrast to astrocytes, the endothelium has been clearly demonstrated to possess abundant COX-1 and PGI₂ synthase enzymes.¹⁴⁰ PGI₂ derived from the activities of these enzymes is the primary vasodilatory PG liberated from the endothelium in response to a variety of stimuli, including agonists of muscarinic, bradykinin and purinergic receptors.^141,142 Similar to the actions of PGE₂ on EP receptors, PGI₂ activates G_s-coupled I prostanoid receptors on SMCs, evoking PKA-mediated vasodilation.¹⁴⁰ Although, PGI₂ and its analogues do relax cerebral arteries and arterioles,¹⁴³ preliminary evidence from mouse brain slices suggests that PGI₂ is not involved in signaling during NVC.¹⁴⁴

The parenchymal endothelium is also a likely source of EETs, as ECs produce these molecules in a range of vascular beds.^145,146 Moreover, cerebral arteries and arterioles dilate in response to direct EET application.^129,147 However, to our knowledge, the endothelial production of these molecules has yet to be directly investigated in parenchymal arterioles. Nonetheless, it is reasonable to speculate that endothelial EETs could influence blood flow by interacting with a range of ion channels in the parenchymal NVU, such as myocyte¹⁰ or astrocytic^22,132 BK channels or endothelial,^17,18 SM^49,19 and astrocytic¹⁶ TRPV4 channels. EET-mediated Ca²⁺ influx through endothelial TRP channels¹⁴⁸ could also contribute to endothelial SK and IK channel activation and membrane hyperpolarization.

Nitric oxide: NVC mediator or modulator?

Another commonly touted Ca²⁺-dependent NVC mediator is nitric oxide (NO). However, the potential contribution of NO is complex and its actual role remains uncertain. NO derived from both neuronal and endothelial sources has been implicated in the control of cerebral blood flow,^149–151 whereas it is generally accepted that astrocytes do not generate NO under normal conditions. NO activates guanylate cyclase, leading to the production of cyclic guanosine monophosphate (cGMP).¹⁵² Cyclic GMP, in turn, is a substrate for PKG, which interacts with multiple downstream effectors. Considering ion channel targets alone, PKG activity can modulate the phosphorylation status of BK channels, leading to an increase in their activity,^153,154 and may act through similar mechanisms to inhibit VDCCs.¹⁵⁵ Cyclic GMP can increase RyR-mediated Ca²⁺ spark frequency,⁵⁷ and NO may also decrease IP₃-mediated Ca²⁺ release through the actions of cGMP and PKG.¹⁵⁶ In the parenchymal NVU, NO from either eNOS or nNOS could interact with guanylate cyclase in both astrocytes¹⁵⁷ and SM¹⁵⁸ to increase the activity of BK channels in these respective cell types. Signaling to SM VDCCs or RyRs would also be predicted to promote vasodilation and increased blood flow. Moreover, because NO-cGMP-PKG signaling has been reported to inhibit TRPV4 channels (alone¹⁵⁹ or when complexed with TRPC1¹⁶⁰ or TRPP2¹⁶¹), endothelial and endfoot Ca²⁺ signaling could also be affected.

Recent work has identified a contribution of eNOS signaling to resting tone in isolated pressurized middle cerebral arteries and parenchymal arterioles,¹²⁴ but whether NO is actively recruited during NVC is controversial.^149,162,163 However, emerging evidence supports the concept of endothelial engagement during NVC through the release of parenchymal factors that increase eNOS activity.^164,165 A study by LeMaistre-Stobart and co-workers¹⁶⁵ reported that astrocytic release of D-serine, in combination with glutamate, may activate endothelial N-methyl D-aspartate (NMDA) receptors, which conduct Na⁺ and Ca²⁺ into the cell. This could then stimulate eNOS, increasing the production of NO and causing vasodilation of arterioles through the actions of NO on the adjacent SMCs.¹⁶⁵ In support of this model, this same group previously demonstrated that endothelial NMDA-receptor activation dilates isolated middle cerebral arteries and brain slice parenchymal arterioles through an eNOS-dependent mechanism.¹⁶⁴ However, the functional expression of NMDA receptors in native parenchymal endothelium is yet to be confirmed.

The next frontier: what are the roles of capillary EC and pericyte ion channels in the control of blood flow in the brain microcirculation?

Capillaries vastly outnumber parenchymal arterioles and are intimately associated with neurons and astrocytes, and are also covered by pericytes. Approximately 90% of capillaries have pericytes,¹⁶⁶ which cover an estimated 37% of the endothelial surface area.¹⁶⁷ This angioarchitecture suggests that capillary ECs and pericytes play an important role in communicating neuronal activity from the capillary bed to upstream arterioles to modulate blood flow into the deep microcirculation. Indeed, recent work from the Attwell laboratory, conducted in brain slices and in vivo, suggested that hyperemia is initiated at the level of pericytes before being conducted upstream to arterioles.¹⁶⁸ These authors demonstrated that brain capillary pericytes are capable of both contraction (in response to noradrenaline) and relaxation in response to exogenous agonists and—importantly—neuronal activity.^168,169 Of particular note, glutamate, NMDA or neuronal stimulation evoked a ∼30 pA hyperpolarizing current in pericytes patch-clamped in situ, thought to underlie pericyte relaxation. The ion channel mediating this hyperpolarization was not firmly identified, although the authors suggest it is highly likely to conduct K⁺.¹⁶⁸ Interestingly, only 25–30% of brain pericytes respond to contractile stimuli,¹⁶⁹ suggesting a sub-population of non-contractile pericytes.

To date ion channel expression and function have not been well explored in brain pericytes, but more is known about retinal pericytes. Relevant in this context, retinal pericytes are known to possess VDCCs,^170,171 endowing them with an avenue through which V_m changes can modulate intracellular Ca²⁺ and adjust the contractile state of the cell. Retinal pericytes also possess K_IR channels,¹⁷² K_Ca (BK and SK) channels^173,174 and, in contrast to parenchymal SM, K_ATP channels.¹⁷⁵ If this complement of K⁺ channels is also present in brain pericytes, they could be harnessed by a variety of signaling pathways – for example external K⁺ activation of K_IR channels – to produce membrane hyperpolarization and relaxation in response to neuronal activity, thereby modulating capillary blood flow (Figure 8). Notably, the ion channel expression profiles of contractile and non-contractile pericytes are likely to differ substantially, reflecting different roles for each subclass of pericytes—an area that requires further exploration. Readers are directed to Hamilton et al.¹⁷⁶ for a more comprehensive review of the literature on pericyte ion channel expression and function.

Figure 8.

Neurovascular communication at the capillary level. Capillary ECs possess K_IR and TRPV4 channels, which can hyperpolarize the membrane and allow Ca²⁺ into the cytosol, respectively. K_IR channels may endow capillary ECs with the ability to sense K⁺ released by neuronal activity and to conduct a regenerative, retrograde hyperpolarization by virtue of the voltage-dependence of K_IR channels. This will lead to rapid upstream dilation of parenchymal arterioles and pial arteries. Pericytes cover more than one-third of the capillary surface area and express VDCCs as a major pathway for the delivery of Ca²⁺ for contraction. They also express K_ATP, K_Ca and K_IR channels, which could be recruited through various mechanisms triggered by neuronal activity to drive membrane hyperpolarization and induce pericyte relaxation. Collectively, ion channels in both ECs and pericytes may provide the means to finely control blood flow deep within the vascular tree.

Less still is known about native ion channels functionally expressed in brain capillary ECs, but preliminary work indicates that these cells possess both K_IR and TRPV4 channels, but unexpectedly lack SK and IK channels and K_ATP channels.¹⁷⁷ This repertoire, although incompletely characterized, equips capillaries with a molecular toolkit for detecting local changes in K⁺ concentration during neuronal activity and responding with robust hyperpolarization; it also provides a pathway for the elevation of intracellular Ca²⁺. Membrane hyperpolarization and propagating intercellular Ca²⁺ waves are prominent features of conducted signaling along arterioles,¹⁷⁸ which is primarily enabled by the endothelial lining.¹⁷⁹ Therefore, capillary K_IR and TRPV4 channels may endow capillaries with the ability to rapidly communicate to upstream arterioles and pericytes—indeed the endothelium has been suggested as an efficient electrical pathway linking pericytes in the retina¹⁸⁰—to finely tune blood flow in the deep microcirculation (Figure 8).

Conclusion

The cells of the NVU express a diverse array of ion channels that engage in intra- and intercellular signaling to control the diameter of parenchymal arterioles and modulate cerebral blood flow. There are close similarities in ion channel expression and function between astrocytic endfeet and ECs, leading to parallels in the signaling mechanisms employed by both cell types to control SM V_m, vascular tone and blood flow. Any defect—for example the accumulation of perivascular deposits such as occurs in Alzheimer’s disease or CADASIL⁶—could impair ion channel signaling during NVC. As the field progresses and the roles of ion channel signaling in the control of cerebral blood flow are more clearly defined, future studies will likely establish NVU ion channel dysregulation and NVC impairment as important mechanisms in a broad range of cerebrovascular disease states.

Footnotes

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article:

This work was supported by the American Heart Association (TAL, award IDs: 12POST12090001, 14POST20480144), the Totman Medical Research Trust (MTN), Fondation Leducq (MTN), and National Institutes of Health (P20-RR-16435, P01-HL-095488, R01-HL-121706, R37-DK-053832 to MTN).

Acknowledgements

The authors gratefully acknowledge Drs. F Dabertrand, T Dalsgaard, A Gonzales and N Villalba for insightful discussions.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

Roy

Sherrington

. On the regulation of the blood-supply of the brain. J Physiol 1890; 11: 85–158.

O'Brien

Eagger

Syed

. A study of regional cerebral blood flow and cognitive performance in Alzheimer's disease. J Neurol Neurosurg Psychiatry 1992; 55: 1182–1187.

Jennings

Muldoon

Ryan

. Reduced cerebral blood flow response and compensation among patients with untreated hypertension. Neurology 2005; 64: 1358–1365.

Prunell

Mathiesen

Svendgaard

N-A

. Experimental subarachnoid hemorrhage: cerebral blood flow and brain metabolism during the acute phase in three different models in the rat. Neurosurgery 2004; 54: 426–437.

Dandona

James

Newbury

. Cerebral blood flow in diabetes mellitus: evidence of abnormal cerebrovascular reactivity. Br Med J 1978; 2: 325–326.

Chabriat

Joutel

Dichgans

. CADASIL. Lancet Neurol 2009; 8: 643–653.

Mishima

Hirase

. In vivo intracellular recording suggests that gray matter astrocytes in mature cerebral cortex and hippocampus are electrophysiologically homogeneous. J Neurosci 2010; 30: 3093–3100.

Emerson

Segal

. Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries: role in vasomotor control. Circ Res 2000; 87: 474–479.

Nystoriak

O'Connor

Sonkusare

. Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization. Am J Physiol Heart Circ Physiol 2011; 300: H803–812.

10.

Hannah

Dunn

Bonev

. Endothelial SK_Ca and IK_Ca channels regulate brain parenchymal arteriolar diameter and cortical cerebral blood flow. J Cereb Blood Flow Metab 2010; 21: 69–78.

11.

Straub

Bonev

Wilkerson

. Dynamic inositol trisphosphate-mediated calcium signals within astrocytic endfeet underlie vasodilation of cerebral arterioles. J Gen Physiol 2006; 128: 659–669.

12.

Ledoux

Taylor

Bonev

. Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections. Proc Natl Acad Sci USA 2008; 105: 9627–9632.

13.

Zhao

Adebiyi

Blaskova

. Type 1 inositol 1,4,5-trisphosphate receptors mediate UTP-induced cation currents, Ca²⁺ signals, and vasoconstriction in cerebral arteries. Am J Physiol Cell Physiol 2008; 295: C1376–1384.

14.

Dabertrand

Nelson

Brayden

. Acidosis dilates brain parenchymal arterioles by conversion of calcium waves to sparks to activate BK channels. Circ Res 2012; 110: 285–294.

15.

Filosa

Bonev

Nelson

. Calcium dynamics in cortical astrocytes and arterioles during neurovascular coupling. Circ Res 2004; 95: e73–81.

16.

Dunn

Hill-Eubanks

Liedtke

. TRPV4 channels stimulate Ca²⁺-induced Ca²⁺ release in astrocytic endfeet and amplify neurovascular coupling responses. Proc Natl Acad Sci USA 2013; 110: 6157–6162.

17.

Marrelli

O'neil

Brown

. PLA2 and TRPV4 channels regulate endothelial calcium in cerebral arteries. Am J Physiol Heart Circ Physiol 2007; 292: H1390–1397.

18.

Zhang

Papadopoulos

Hamel

. Endothelial TRPV4 channels mediate dilation of cerebral arteries: impairment and recovery in cerebrovascular pathologies related to Alzheimer disease. Br J Pharmacol 2013; 170: 661–670.

19.

Baylie

Tavares

Navedo

. The role of TRPV4 in rat parenchymal arterioles. FASEB J 2010. 1033.2 (abstract).

20.

Baylie

Tavares

. TRPM4 channels couple purinergic receptor mechanoactivation and myogenic tone development in cerebral parenchymal arterioles. J Cereb Blood Flow Metab 2014; 34: 1706–1714.

21.

Gonzales

Yang

Sullivan

. A PLCγ1-dependent, force-sensitive signaling network in the myogenic constriction of cerebral arteries. Sci Signal 2014; 7: ra49.

22.

Filosa

Bonev

Straub

. Local potassium signaling couples neuronal activity to vasodilation in the brain. Nat Neurosci 2006; 9: 1397–1403.

23.

Girouard

Bonev

Hannah

. Astrocytic endfoot Ca²⁺ and BK channels determine both arteriolar dilation and constriction. Proc Natl Acad Sci USA 2010; 107: 3811–3816.

24.

Longden

Dunn

Draheim

. Intermediate-conductance calcium-activated potassium channels participate in neurovascular coupling. Br J Pharmacol 2011; 164: 922–933.

25.

Straub

Girouard

Doetsch

. Regulation of intracerebral arteriolar tone by K_v channels: effects of glucose and PKC. Am J Physiol Cell Physiol 2009; 297: C788–796.

26.

Longden

Nelson

. Vascular inward rectifier K⁺ channels as external K⁺ sensors in the control of cerebral blood flow. Microcirculation 2015; 22: 183–196.

27.

Crane

Walker

Dora

. Evidence for a differential cellular distribution of inward rectifier K channels in the rat isolated mesenteric artery. J Vasc Res 2003; 40: 159–168.

28.

Longden

Dabertrand

Hill-Eubanks

. Stress-induced glucocorticoid signaling remodels neurovascular coupling through impairment of cerebrovascular inwardly rectifying K⁺ channel function. Proc Natl Acad Sci USA 2014; 111: 7462–7467.

29.

Zaritsky

Eckman

Wellman

. Targeted disruption of Kir2.1 and Kir2.2 genes reveals the essential role of the inwardly rectifying K⁺ current in K⁺-mediated vasodilation. Circ Res 2000; 87: 160–166.

30.

Cipolla

. The cerebral circulation, San Rafael, CA: Morgan & Claypool Life Sciences, 2009.

31.

Simard

Arcuino

Takano

. Signaling at the gliovascular interface. J Neurosci 2003; 23: 9254–9262.

32.

Nishimura

Schaffer

Friedman

. Penetrating arterioles are a bottleneck in the perfusion of neocortex. Proc Natl Acad Sci USA 2007; 104: 365–370.

33.

Aydin

Rosenblum

Povlishock

. Myoendothelial junctions in human brain arterioles. Stroke 1991; 22: 1592–1597.

34.

Iliff

Wang

Liao

. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including Amyloid β. Sci Transl Med 2012; 4: 147ra111.

35.

Brown

Davies

Speake

. Molecular mechanisms of cerebrospinal fluid production. Neuroscience 2004; 129: 955–968.

36.

Knot

Nelson

. Regulation of arterial diameter and wall [Ca²⁺] in cerebral arteries of rat by membrane potential and intravascular pressure. J Physiol 1998; 508: 199–209.

37.

Bayliss

. On the local reactions of the arterial wall to changes of internal pressure. J Physiol 1902; 28: 220–231.

38.

Nelson

Patlak

Worley

. Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone. Am J Physiol 1990; 259: C3–18.

39.

Catterall

. Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 2000; 16: 521–555.

40.

Kuo

Ellis

Seymour

. Dihydropyridine-insensitive calcium currents contribute to function of small cerebral arteries. J Cereb Blood Flow Metab 2010; 30: 1226–1239.

41.

Abd El-Rahman

Harraz

Brett

. Identification of L- and T-type Ca²⁺ channels in rat cerebral arteries: role in myogenic tone development. Am J Physiol Heart Circ Physiol 2013; 304: H58–H71.

42.

Nelson

Quayle

. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 1995; 268: C799–C822.

43.

Brayden

Earley

Nelson

. Transient receptor potential (TRP) channels, vascular tone and autoregulation of cerebral blood flow. Clin Exp Pharmacol Physiol 2008; 35: 1116–1120.

44.

Ramsey

Delling

Clapham

. An introduction to TRP channels. Annu Rev Physiol 2006; 68: 619–647.

45.

Earley

Brayden

. Transient receptor potential channels in the vasculature. Physiol Rev 2015; 95: 645–690.

46.

Earley

Waldron

Brayden

. Critical role for transient receptor potential channel TRPM4 in myogenic constriction of cerebral arteries. Circ Res 2004; 95: 922–929.

47.

Welsh

Morielli

Nelson

. Transient receptor potential channels regulate myogenic tone of resistance arteries. Circ Res 2002; 90: 248–250.

48.

Adebiyi

Zhao

. IP₃ constricts cerebral arteries via IP₃ receptor–mediated TRPC3 channel activation and independently of sarcoplasmic reticulum Ca²⁺ release. Circ Res 2008; 102: 1118–1126.

49.

Earley

Heppner

Nelson

. TRPV4 forms a novel Ca²⁺ signaling complex with ryanodine receptors and BK_Ca channels. Circ Res 2005; 97: 1270–1279.

50.

Gonzales

Amberg

Earley

. Ca²⁺ release from the sarcoplasmic reticulum is required for sustained TRPM4 activity in cerebral artery smooth muscle cells. Am J Physiol Cell Physiol 2010; 299: C279–288.

51.

Gonzales

Garcia

Amberg

. Pharmacological inhibition of TRPM4 hyperpolarizes vascular smooth muscle. Am J Physiol Cell Physiol 2010; 299: C1195–1202.

52.

Dietrich

Gudermann

TRPC6. In: Flockerzi

Nilius

(eds). Transient receptor potential (TRP) channels, Heidelberg: Springer, 2007, pp. 125–142.

53.

Maroto

Raso

Wood

. TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nat Cell Biol 2005; 7: 179–185.

54.

Becker

Bereiter-Hahn

Jendrach

. Functional interaction of the cation channel transient receptor potential vanilloid 4 (TRPV4) and actin in volume regulation. Eur J Cell Biol 2009; 88: 141–152.

55.

Köhler

Heyken

Heinau

. Evidence for a functional role of endothelial transient receptor potential V4 in shear stress-induced vasodilatation. Arterioscler Thromb Vasc Biol 2006; 26: 1495–1502.

56.

Hill-Eubanks

Gonzales

Sonkusare

. Vascular TRP Channels: Performing Under Pressure and Going with the Flow. Physiology 2014; 29: 343–360.

57.

Porter

Bonev

Knot

. Frequency modulation of Ca²⁺ sparks is involved in regulation of arterial diameter by cyclic nucleotides. Am J Physiol 1998; 274: C1346–1355.

58.

Brayden

Tavares

. Purinergic receptors regulate myogenic tone in cerebral parenchymal arterioles. J Cereb Blood Flow Metab 2013; 33: 293–299.

59.

Nicholas

Watt

Lazarowski

. Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor. Mol Pharmacol 1996; 50: 224–229.

60.

Baumbach

Sigmund

Faraci

. Cerebral arteriolar structure in mice overexpressing human renin and angiotensinogen. Hypertension 2003; 41: 50–55.

61.

Nelson

Cheng

Rubart

. Relaxation of arterial smooth muscle by calcium sparks. Science 1995; 270: 633–637.

62.

Lanner

Georgiou

Joshi

. Ryanodine receptors: structure, expression, molecular details, and function in calcium release. Cold Spring Harb Perspect Biol 2010; 2: a003996.

63.

Mufti

Brett

Tran

CHT

. Intravascular pressure augments cerebral arterial constriction by inducing voltage-insensitive Ca²⁺ waves. J Physiol 2010; 588: 3983–4005.

64.

Jaggar

Porter

Lederer

. Calcium sparks in smooth muscle. Am J Physiol Cell Physiol 2000; 278: C235–256.

65.

Peréz

Bonev

Nelson

. Micromolar Ca²⁺ from sparks activates Ca^2+-sensitive K⁺ channels in rat cerebral artery smooth muscle. Am J Physiol Cell Physiol 2001; 281: C1769–1775.

66.

Heppner

Bonev

Santana

. Alkaline pH shifts Ca²⁺ sparks to Ca²⁺ waves in smooth muscle cells of pressurized cerebral arteries. Am J Physiol Heart Circ Physiol 2002; 283: H2169–2176.

67.

Brayden

Nelson

. Regulation of arterial tone by activation of calcium-dependent potassium channels. Science 1992; 256: 532–535.

68.

Dabertrand

Nelson

Brayden

. Ryanodine receptors, calcium signaling, and regulation of vascular tone in the cerebral parenchymal microcirculation. Microcirculation 2013; 20: 307–316.

69.

Foskett

White

Cheung

. Inositol trisphosphate receptor Ca²⁺ release channels. Physiol Rev 2007; 87: 593–658.

70.

Reading

Earley

Waldron

. TRPC3 mediates pyrimidine receptor-induced depolarization of cerebral arteries. Am J Physiol Heart Circ Physiol 2005; 288: H2055–2061.

71.

Gomez

Stevenson

Bonev

. Opposing actions of Inositol 1,4,5-Trisphosphate and ryanodine receptors on nuclear factor of activated T-cells regulation in smooth muscle. J Biol Chem 2002; 277: 37756–37764.

72.

Hibino

Inanobe

Furutani

. Inwardly rectifying potassium channels: Their structure, function, and physiological roles. Physiol Rev 2010; 90: 291–366.

73.

Quayle

McCarron

Brayden

. Inward rectifier K⁺ currents in smooth muscle cells from rat resistance-sized cerebral arteries. Am J Physiol Cell Physiol 1993; 265: C1363–1370.

74.

Lopatin

Makhina

Nichols

. Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature 1994; 372: 366–369.

75.

Jansen-Olesen

Mortensen

El-Bariaki

. Characterization of K_ATP-channels in rat basilar and middle cerebral arteries: studies of vasomotor responses and mRNA expression. Eur J Pharmacol 2005; 523: 109–118.

76.

Armstead

. Relationship among NO, the K_ATP channel, and opioids in hypoxic pial artery dilation. Am J Physiol Heart Circ Physiol 1998; 275: H988–994.

77.

Lindauer

Vogt

Schuh-Hofer

. Cerebrovascular vasodilation to extraluminal acidosis occurs via combined activation of ATP-sensitive and Ca²⁺-activated potassium channels. J Cereb Blood Flow Metab 2003; 23: 1227–1238.

78.

Wei

Gutman

Aldrich

. International Union of Pharmacology. LII. Nomenclature and molecular relationships of calcium-activated potassium channels. Pharmacol Rev 2005; 57: 463–472.

79.

Ledoux

Werner

Brayden

. Calcium-activated potassium Channels and the regulation of vascular tone. Physiology 2006; 21: 69–78.

80.

Gutman

Chandy

Grissmer

. International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels. Pharmacoll Rev 2005; 57: 473–508.

81.

Long

Campbell

Mackinnon

. Crystal structure of a mammalian voltage-dependent Shaker family K⁺ channel. Science 2005; 309: 897–903.

82.

Albarwani

Nemetz

Madden

. Voltage-gated K⁺ channels in rat small cerebral arteries: molecular identity of the functional channels. J Physiol 2009; 551: 751–763.

83.

Dabertrand

Krøigaard

Bonev

. Potassium channelopathy-like defect underlies early-stage cerebrovascular dysfunction in a genetic model of small vessel disease. Proc Natl Acad Sci USA 2015; 112: E796–805.

84.

Wallraff

Odermatt

Willecke

. Distinct types of astroglial cells in the hippocampus differ in gap junction coupling. Glia 2004; 48: 36–43.

85.

Tang

Taniguchi

Kofuji

. Heterogeneity of Kir4.1 channel expression in glia revealed by mouse transgenesis. Glia 2009; 57: 1706–1715.

86.

Zhou

Schools

Kimelberg

. Development of GLAST⁺ astrocytes and NG2⁺ glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive. J Neurophysiol 2005; 95: 134–143.

87.

Emerson

Segal

. Electrical activation of endothelium evokes vasodilation and hyperpolarization along hamster feed arteries. Am J Physiol Heart Circ Physiol 2001; 280: H160–167.

88.

Yamamoto

Imaeda

Suzuki

. Endothelium-dependent hyperpolarization and intercellular electrical coupling in guinea-pig mesenteric arterioles. J Physiol 1999; 514: 505–513.

89.

Dora

Xia

Duling

. Endothelial cell signaling during conducted vasomotor responses. Am J Physiol Heart Circ Physiol 2003; 285: H119–126.

90.

Sonkusare

Bonev

Ledoux

. Elementary Ca²⁺ signals through endothelial TRPV4 channels regulate vascular function. Science 2012; 336: 597–601.

91.

Porter

McCarthy

. Astrocytic neurotransmitter receptors in situ and in vivo. Prog Neurobiol 1997; 51: 439–455.

92.

van den Pol

Romano

Ghosh

. Metabotropic glutamate receptor mGluR5 subcellular distribution and developmental expression in hypothalamus. J Comp Neurol 1995; 362: 134–150.

93.

Agulhon

Petravicz

McMullen

. What is the role of astrocyte calcium in neurophysiology? Neuron 2008; 59: 932–946.

94.

Petravicz

Fiacco

McCarthy

. Loss of IP₃ receptor-dependent Ca²⁺ increases in hippocampal astrocytes does not affect baseline CA1 pyramidal neuron synaptic activity. J Neurosci 2008; 28: 4967–4973.

95.

Linden

Sapirstein

. Astrocyte inositol triphosphate receptor type 2 and cytosolic phospholipase A2 alpha regulate arteriole responses in mouse neocortical brain slices. PLoS ONE 2012; 7: e42194.

96.

Takata

Mishima

Hisatsune

. Astrocyte calcium signaling transforms cholinergic modulation to cortical plasticity in vivo. J Neurosci 2011; 31: 18155–18165.

97.

Takata

Nagai

Ozawa

. Cerebral blood flow modulation by basal forebrain or whisker stimulation can occur independently of large cytosolic Ca²⁺ signaling in astrocytes. PLoS ONE 2013; 8: e66525.

98.

Nizar

Uhlirova

Tian

. In vivo stimulus-induced vasodilation occurs without IP₃ receptor activation and may precede astrocytic calcium increase. J Neurosci 2013; 33: 8411–8422.

99.

Takano

Tian

G-F

Peng

. Astrocyte-mediated control of cerebral blood flow. Nat Neurosci 2005; 9: 260–267.

100.

Mulligan

MacVicar

. Calcium transients in astrocyte endfeet cause cerebrovascular constrictions. Nature 2004; 431: 195–199.

101.

Koide

Bonev

Nelson

. Inversion of neurovascular coupling by subarachnoid blood depends on large-conductance Ca2⁺-activated K⁺ (BK) channels. Proc Natl Acad Sci USA 2012; 109: E1387–1395.

102.

Srinivasan

Huang

Venugopal

. Ca²⁺ signaling in astrocytes from Ip3r2^−/− mice in brain slices and during startle responses in vivo. Nat Neurosci 2015; 18: 708–717.

103.

Otsu

Couchman

Lyons

. Calcium dynamics in astrocyte processes during neurovascular coupling. Nat Neurosci 2015; 18: 210–218.

104.

Lind

Brazhe

Jessen

. Rapid stimulus-evoked astrocyte Ca²⁺ elevations and hemodynamic responses in mouse somatosensory cortex in vivo. Proc Natl Acad Sci USA 2013; 110: E4678–4687.

105.

Zonta

Angulo

Gobbo

. Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nat Neurosci 2003; 6: 43–50.

106.

Pasti

Volterra

Pozzan

. Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ. J Neurosci 1997; 17: 7817–7830.

107.

Sun

McConnell

Pare

. Glutamate-dependent neuroglial calcium signaling differs between young and adult brain. Science 2013; 339: 197–200.

108.

Wang

Lou

. Astrocytic Ca²⁺ signaling evoked by sensory stimulation in vivo. Nat Neurosci 2006; 9: 816–823.

109.

Chen

Kozberg

Bouchard

. A critical role for the vascular endothelium in functional neurovascular coupling in the brain. J Am Heart Assoc 2014; 3: e000787.

110.

Edwards

Félétou

Weston

. Endothelium-derived hyperpolarising factors and associated pathways: a synopsis. Pflugers Arch 2010; 459: 863–879.

111.

Benfenati

Amiry-Moghaddam

Caprini

. Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in rat cortical astrocytes. Neuroscience 2007; 148: 876–892.

112.

O'Neil

Heller

. The mechanosensitive nature of TRPV channels. Pflugers Arch 2005; 451: 193–203.

113.

Filosa

Iddings

. Astrocyte regulation of cerebral vascular tone. Am J Physiol Heart Circ Physiol 2013; 305: H609–619.

114.

Moore

Cao

. The hemo-neural hypothesis: on the role of blood flow in information processing. J Neurophysiol 2008; 99: 2035–2047.

115.

Sonkusare

Dalsgaard

Bonev

. AKAP150-dependent cooperative TRPV4 channel gating is central to endothelium dependent vasodilation and is disrupted in hypertension. Sci Signal 2014; 7: ra66.

116.

Bagher

Beleznai

Kansui

. Low intravascular pressure activates endothelial cell TRPV4 channels, local Ca²⁺ events, and IK_Ca channels, reducing arteriolar tone. Proc Natl Acad Sci USA 2012; 109: 18174–18179.

117.

Sonkusare

Villalba

Freeman

. Cooperative gating and sensitivity of TRPV4 channels are regulated by distinct factors. FASEB J 2014. 1057.8 (abstract).

118.

Price

Ludwig

. Distribution of rSlo Ca²⁺-activated K⁺ channels in rat astrocyte perivascular endfeet. Brain Res 2002; 956: 183–193.

119.

Witthoft

Karniadakis

. A bidirectional model for communication in the neurovascular unit. J Theor Biol 2012; 311: 80–93.

120.

Witthoft

Filosa

Karniadakis

. Potassium buffering in the neurovascular unit: models and sensitivity analysis. Biophys J 2013; 105: 2046–2054.

121.

Farr

David

. Models of neurovascular coupling via potassium and EET signalling. J Theor Biol 2011; 286: 13–23.

122.

Xia

Fakler

Rivard

. Mechanism of calcium gating in small-conductance calcium-activated potassium channels. Nature 1998; 395: 503–507.

123.

Köhler

Hirschberg

Bond

. Small-conductance, calcium-activated potassium channels from mammalian brain. Science 1996; 273: 1709–1714.

124.

Cipolla

Smith

Kohlmeyer

. SK_Ca and IK_Ca channels, myogenic tone, and vasodilator responses in middle cerebral arteries and parenchymal arterioles: Effect of ischemia and reperfusion. Stroke 2009; 40: 1451–1457.

125.

Dora

Gallagher

McNeish

. Modulation of endothelial cell K_Ca3.1 channels during endothelium-derived hyperpolarizing factor signaling in mesenteric resistance arteries. Circ Res 2008; 102: 1247–1255.

126.

Taylor

Bonev

Gross

. Altered expression of small-conductance Ca²⁺-activated K⁺ (SK3) channels modulates arterial tone and blood pressure. Circ Res 2003; 93: 124–131.

127.

Burnham

Bychkov

Feletou

. Characterization of an apamin-sensitive small-conductance Ca²⁺-activated K⁺ channel in porcine coronary artery endothelium: relevance to EDHF. Br J Pharmacol 2002; 135: 1133–1143.

128.

Harder

Alkayed

Lange

. Functional hyperemia in the brain: hypothesis for astrocyte-derived vasodilator metabolites. Stroke 1998; 29: 229–234.

129.

Gebremedhin

Falck

. Mechanism of action of cerebral epoxyeicosatrienoic acids on cerebral arterial smooth muscle. Am J Physiol 1992; 263: H519–525.

130.

Peng

Zhang

Alkayed

. Dependency of cortical functional hyperemia on epoxygenase and nitric oxide synthase activities in rats. J Cereb Blood Flow Metab 2004; 24: 509–517.

131.

Alkayed

Narayanan

Gebremedhin

. Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes. Stroke 1996; 27: 971–979.

132.

Higashimori

Blanco

Tuniki

. Role of epoxyeicosatrienoic acids as autocrine metabolites in glutamate-mediated K⁺ signaling in perivascular astrocytes. Am J Physiol Cell Physiology 2010; 299: C1068–1078.

133.

Campbell

Gebremedhin

Pratt

. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res 1996; 78: 415–423.

134.

Chen

Bortell

. 11,12-Epoxyeicosatrienoic acid stimulates endogenous mono-ADP-ribosylation in bovine coronary arterial smooth muscle. Circ Res 1999; 85: 349–356.

135.

Attwell

Buchan

Charpak

. Glial and neuronal control of brain blood flow. Nature 2010; 468: 232–243.

136.

Gordon

GRJ

Choi

Rungta

. Brain metabolism dictates the polarity of astrocyte control over arterioles. Nature 2008; 456: 745–749.

137.

Lecrux

Toussay

Kocharyan

. Pyramidal neurons are ‘neurogenic hubs’ in the neurovascular coupling response to whisker stimulation. J Neurosci 2011; 31: 9836–9847.

138.

Lacroix

Toussay

Anenberg

. COX-2-derived prostaglandin E₂ produced by pyramidal neurons contributes to neurovascular coupling in the rodent cerebral cortex. J Neurosci 2015; 35: 11791–11810.

139.

Dabertrand

Hannah

Pearson

. Prostaglandin E2, a postulated astrocyte-derived neurovascular coupling agent, constricts rather than dilates parenchymal arterioles. J Cereb Blood Flow Metab 2013; 33: 479–482.

140.

Félétou

. The endothelium: Part 1: Multiple functions of the endothelial cells—focus on endothelium-derived vasoactive mediators, San Rafael, CA: Morgan & Claypool Life Sciences, 2011.

141.

Bunting

Gryglewski

Moncada

. Arterial walls generate from prostaglandin endoperoxides a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac ateries and inhibits platelet aggregation. Prostaglandins 1976; 12: 897–913.

142.

Moncada

Gryglewski

Bunting

. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 1976; 263: 663–665.

143.

Ellis

Wei

Kontos

. Vasodilation of cat cerebral arterioles by prostaglandins D2, E2, G2, and I2. Am J Physiol 1979; 237: H381–385.

144.

Longden

Nelson

. Recruitment of the vascular endothelium into neurovascular coupling. FASEB J 2012; 26. 842.4 (abstract).

145.

Archer

Gragasin

. Endothelium-derived hyperpolarizing factor in human internal mammary artery is 11,12-epoxyeicosatrienoic acid and causes relaxation by activating smooth muscle BK_Ca channels. Circulation 2003; 107: 769–776.

146.

Campbell

Falck

Gauthier

. Role of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factor in bovine coronary arteries. Med Sci Monit 2001; 7: 578–584.

147.

Ellis

Police

Yancey

. Dilation of cerebral arterioles by cytochrome P-450 metabolites of arachidonic acid. Am J Physiol 1990; 259: H1171–1177.

148.

Fleming

Rueben

Popp

. Epoxyeicosatrienoic acids regulate Trp channel dependent Ca²⁺ signaling and hyperpolarization in endothelial cells. Arterioscler Thromb Vasc Biol 2007; 27: 2612–2618.

149.

De Labra

Rivadulla

Espinosa

. Different sources of nitric oxide mediate neurovascular coupling in the lateral geniculate nucleus of the cat. Front Syst Neurosci 2009; 3: 9.

150.

Iadecola

Zhang

. Role of nitric oxide synthase-containing vascular nerves in cerebrovasodilation elicited from cerebellum. Am J Physiol 1993; 264: R738–746.

151.

Kitaura

Uozumi

Tohmi

. Roles of nitric oxide as a vasodilator in neurovascular coupling of mouse somatosensory cortex. Neurosci Res 2007; 59: 160–171.

152.

Denninger

Marletta

. Guanylate cyclase and the.NO/cGMP signaling pathway. Biochim Biophys Acta 1999; 1411: 334–350.

153.

Archer

Huang

Hampl

. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci USA 1994; 91: 7583–7587.

154.

White

Lee

Shcherbatko

. Potassium channel stimulation by natriuretic peptides through cGMP-dependent dephosphorylation. Nature 1993; 361: 263–266.

155.

Yang

Liu

Zakharov

. Protein kinase G phosphorylates Cav1.2 1c and 2 subunits. Circ Res 2007; 101: 465–474.

156.

Schlossmann

Ammendola

Ashman

. Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta. Nature 2000; 404: 197–201.

157.

Teunissen

Steinbusch

Markerink-van Ittersum

. Presence of soluble and particulate guanylyl cyclase in the same hippocampal astrocytes. Brain Res 2001; 891: 206–212.

158.

Sobey

Faraci

. Effects of a novel inhibitor of guanylyl cyclase on dilator responses of mouse cerebral arterioles. Stroke 1997; 28: 837–842.

159.

Yin

Hoffmann

Kaestle

. Negative-feedback loop attenuates hydrostatic lung edema via a cGMP-dependent regulation of transient receptor potential vanilloid 4. Circ Res 2008; 102: 966–974.

160.

Qiu

Luo

. Functional role of vanilloid transient receptor potential 4-canonical transient receptor potential 1 complex in flow-induced Ca²⁺ influx. Arterioscler Thromb Vasc Biol 2010; 30: 851–858.

161.

Wong

Sun

. Protein kinase G inhibits flow-induced Ca²⁺ entry into collecting duct cells. J Am Soc Nephrol 2012; 23: 1172–1180.

162.

Lindauer

Megow

Matsuda

. Nitric oxide: a modulator, but not a mediator, of neurovascular coupling in rat somatosensory cortex. Am J Physiol Heart Circ Physiol 1999; 277: H799–811.

163.

Yang

Iadecola

. Obligatory role of NO in glutamate-dependent hyperemia evoked from cerebellar parallel fibers. Am J Physiol 1997; 272: R1155–1161.

164.

LeMaistre

Sanders

Stobart

. Coactivation of NMDA receptors by glutamate and D-serine induces dilation of isolated middle cerebral arteries. J Cereb Blood Flow Metab 2011; 32: 537–547.

165.

Stobart

JLL

Anderson

HDI

. Astrocyte-induced cortical vasodilation is mediated by D-serine and endothelial nitric oxide synthase. Proc Natl Acad Sci USA 2013; 110: 3149–3154.

166.

Zechariah

ElAli

Doeppner

. Vascular endothelial growth factor promotes pericyte coverage of brain capillaries, improves cerebral blood flow during subsequent focal cerebral ischemia, and preserves the metabolic penumbra. Stroke 2013; 44: 1690–1697.

167.

Mathiisen

Lehre

Danbolt

. The perivascular astroglial sheath provides a complete covering of the brain microvessels: an electron microscopic 3D reconstruction. Glia 2010; 58: 1094–1103.

168.

Hall

Reynell

Gesslein

. Capillary pericytes regulate cerebral blood flow in health and disease. Nature 2014; 508: 55–60.

169.

Peppiatt

Howarth

Mobbs

. Bidirectional control of CNS capillary diameter by pericytes. Nature 2006; 443: 700–704.

170.

Sugiyama

Kawamura

Yamanishi

. Regulation of P2X7-induced pore formation and cell death in pericyte-containing retinal microvessels. Am J Physiol Cell Physiol 2005; 288: C568–576.

171.

Kawamura

Kobayashi

. Effects of angiotensin II on the pericyte-containing microvasculature of the rat retina. J Physiol 2004; 561: 671–683.

172.

Matsushita

Puro

. Topographical heterogeneity of K_IR currents in pericyte-containing microvessels of the rat retina: effect of diabetes. J Physiol 2006; 573: 483–495.

173.

Quignard

Harley

Duhault

. K⁺ channels in cultured bovine retinal pericytes: effects of beta-adrenergic stimulation. J Cardiovasc Pharmacol 2003; 42: 379–388.

174.

Wiederholt

Berweck

Helbig

. Electrophysiological properties of cultured retinal capillary pericytes. Prog Retin Eye Res 1995; 14: 437–451.

175.

Puro

. Adenosine activates ATP-sensitive K⁺ currents in pericytes of rat retinal microvessels: role of A1 and A2a receptors. Brain Res 2001; 907: 93–99.

176.

Hamilton

Attwell

Hall

. Pericyte-mediated regulation of capillary diameter: a component of neurovascular coupling in health and disease. Front Neuroenerg 2010; 2: 5.

177.

Longden

Nelson

. Unique ion channel properties of brain capillary endothelial cells. FASEB J 2015. 832.9 (abstract).

178.

Tallini

Brekke

Shui

. Propagated endothelial Ca²⁺ waves and arteriolar dilation in vivo: Measurements in Cx40 BAC GCaMP2 transgenic mice. Circ Res 2007; 101: 1300–1309.

179.

Looft-Wilson

Payne

Segal

. Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle. J Appl Physiol 2004; 97: 1152–1158.

180.

Minami

Kawamura

. Electrotonic transmission within pericyte-containing retinal microvessels. Microcirculation 2006; 13: 353–363.

Ion channel networks in the control of cerebral blood flow

Abstract

Keywords

Introduction

The neurovascular unit

The ionic composition in the NVU establishes the basal conditions for controlling cerebral blood flow

Part I: control of the smooth muscle membrane potential is central to the control of cerebral blood flow

The key role of the voltage-dependent Ca2+ channel as a membrane potential biosensor

A role for smooth muscle transient receptor potential channels in myogenic depolarization: evidence from pial and parenchymal arterioles

Ryanodine receptors in smooth muscle: Ca2+-release channels that oppose myogenic constriction

IP3 receptors

K+ channels in smooth muscle

Smooth muscle inwardly rectifying K+ channels: Targets of endfoot K+ release

Ca2+-activated K+ channels: the hyperpolarizing influence of smooth muscle BK channel activity can be exploited by NVC mediators

KV channels exert a tonic hyperpolarizing influence on the smooth muscle membrane potential

Part II: dual control of arteriolar smooth muscle by the endothelium and astrocytic endfeet

IP3Rs and TRPV4 play central roles in Ca2+ signaling in the endothelium and endfoot

IP3Rs

TRPV4 channels

Expression of Ca2+ targets in astrocytic endfeet and the endothelium

KCa channels and K+ signaling: a mechanism for bi-directional control of parenchymal arteriole diameter

Arachidonic acid metabolites

Nitric oxide: NVC mediator or modulator?

The next frontier: what are the roles of capillary EC and pericyte ion channels in the control of blood flow in the brain microcirculation?

Conclusion

Footnotes

Funding

Acknowledgements

Declaration of conflicting interests

References

The key role of the voltage-dependent Ca²⁺ channel as a membrane potential biosensor

Ryanodine receptors in smooth muscle: Ca²⁺-release channels that oppose myogenic constriction

IP₃ receptors

K⁺ channels in smooth muscle

Smooth muscle inwardly rectifying K⁺ channels: Targets of endfoot K⁺ release

Ca²⁺-activated K⁺ channels: the hyperpolarizing influence of smooth muscle BK channel activity can be exploited by NVC mediators

K_V channels exert a tonic hyperpolarizing influence on the smooth muscle membrane potential

IP₃Rs and TRPV4 play central roles in Ca²⁺ signaling in the endothelium and endfoot

IP₃Rs

Expression of Ca²⁺ targets in astrocytic endfeet and the endothelium

K_Ca channels and K⁺ signaling: a mechanism for bi-directional control of parenchymal arteriole diameter